1. Field of the Invention
The present invention relates to the analysis of blood formulae by counting and discriminating between leukocyte sub-populations, and more particularly to a reagent and a method of using the same for determining, using flow cytometry, at least one leukocyte sub-population.
2. Description of Related Art
The importance, in making diagnoses, of precisely determining the different leukocyte populations has long been recognized. Indeed, the occurrence of abnormal leukocyte ratios can be correlated with that of certain diseases (immune responses, inflammatory reactions, the appearance of neoplasms or leukaemias, etc.).
The traditional methods of manual analysis, including physically separating the erythrocytes (by sedimentation or aggregation) and then differentially staining the cytoplasms and nuclei of the leukocytes and observing their morphology under the microscope, already permitted identification of all the types of leukocytes, i.e. the granulocytes (basophils, eosinophils and neutrophils and the agranulocytes (monocytes and lymphocytes). These methods yield reliable results, but they are time consuming, which makes them seldom transposable for determinations using automatic apparatus, or only with difficulty.
Different apparatus permitting automatic counting of leukocyte populations are already on the market, and various reagents and staining agents have been developed and adapted to suit the operating principles of such apparatus.
It is possible, for instance, to measure the sizes of the cells (after differential lysis of their cytoplasms), either by measuring variations in resistivity or by measuring optical diffraction; it is also possible to carry out specific staining (enzymatic or otherwise) of different cell types and measure the sizes and optical densities of the cells at different wavelengths.
One of the problems posed by automation is the possibility of confusion between erythrocytes and certain small-sized leukocytes, which is an artefact that did not occur with microscopic observation. Total lysis of the erythrocytes thus has to be ensured before the blood sample is put through the apparatus, at the same time preserving the integrity of the leukocytes. In addition, the reaction mixture has to include one or more reagents that will facilitate discrimination between the different types of leukocytes and their grouping into quite distinct zones on a histogram.
Patent WO 8505684 (Coulter Electronics Inc.) thus describes a group of reagents which ensure, in two stages, the specific lysis of the erythrocytes by saponin, and then the fixation (and thereby protection) of the leukocytes by glutaraldehyde. In addition, discrimination between the different types of leukocytes is favoured by the addition of a reagent such as phenoxy-2-ethanol; certain populations can also be specifically stained and identified by fluorescence. This efficient, reliable, but complex, process is particularly suitable for an apparatus of the Coulter Counter.RTM. type, as described in U.S. Pat. No. 3,502,974.
Other methods, described, for example, in U.S. Pat. No. 3,740,143, which are particularly suitable for Technicon.RTM. apparatus, involve several reactions conducted in parallel, each revealing a cell type, these specific reactions being preceded or followed, moreover, by an erythrocyte lysis treatment, and possibly a leukocyte fixation treatment. The specific reactions include conventional histological staining (such as "neutral red" for the basophilic cells) and enzymatic staining (such as revealing the peroxidase content of the eosinophilic and neutrophil cells using chloronaphthol, or the lipase content of the monocytes using naphthol butyrate).
Generally speaking, detailed, reliable analyses can only be carried out using highly complex apparatus and methods that comprise several different stages, conducted using different reagents. Owing to their complexity, the cost of such analyses is very high and, for the purposes of routine diagnoses, it is always desirable to simplify the process, while preserving the same discrimination power and the same degree of reliability.